Extracts of milk thistle (Silybum marianum) have been used since antiquity for a number of disorders and have most recently been investigated for utility in hepatic disorders of viral and chemical origin, dyslipidemia, and cancer. Milk thistle extracts, often referred to interchangably as silymarin or silibinin, contain a number of flavonoid and flavonolignan compounds that are responsible for their in vitro and in vivo biological effects. However, studies to ascribe particular biological actions to specific milk thistle components have been few in number due both to the complex stereochemistry of the naturally-occurring compounds and the considerable challenges in isolating these compounds in sufficient quantities. The RTI Natural Products Laboratory has recently succeeded in identifying and isolating from commercially-available silymarin the individual stereoand regio-isomers of milk thistle flavonolignans. These compounds, referred to as silybin A, silybin B, isosilybin A, and isosilybin B, are now available for the first time for characterization of their individual biological activities and, specifically, the determination of their relative contribution to the substantial activity of silibinin and silymarin observed previously in pre-clinical models of human prostate cancer. To do so, the group has established a collaboration with the laboratory of Dr. Rajesh Agarwal at the University of Colorado, the international leader in the demonstrating the efficacy of milk thistle extracts in skin and prostate cancer models and the biochemical mechanisms underlying these responses. This collaborative research group proposes to test the HYPOTHESIS that individual flavonolignan isomers possess distinct biological activities that account for the collective anti-cancer action previously observed for the naturally occurring milk thistle extract mixtures, silibinin and silymarin. Specifically, the group proposes 1) to conduct and optimize the semi-synthesis of the 4 milk thistle flavonolignans in order to isolate each in the larger quantities required for all in vitro and in vivo studies, 2) to investigate the antitumor activity of each individual flavonolignan relative to silibinin and silymarin in DU145 hormone-refractory, metastatic human prostate cancer in immunocompromised mice, 3) to determine the specific biological actions of each flavonolignan on in vitro endpoints of mitogenic cellular signaling and cell cycle progression, and 4) to determine the specific biological actions of each flavonolignan on a) genetic elements in the IGFBP-3 and topoisomerase II gene promoters and b) newly described causative/prognostic factors in human prostate cancer (CXCR4, EZH2, and AMACR). Taken together, these studies are anticipated to advance our biochemical understanding of a highly non-toxic prostate cancer prevention and treatment intervention with the possibility of identifying more efficacious and/or bioavailable combinations of compounds than those occurring naturally.